The development of hemoglobin based blood substitutes continues to command commercial attention, and recent developments have shown that hemoglobin from mammalian blood cells, after suitable modification such as intramolecular crosslinking and in some instances polymerization, shows great promise as the basis of a blood substitute. As development has proceeded, however, the requirements for purity of the hemoglobin have steadily increased. At one time, it was believed that hemoglobin simply needed to be stroma free, a condition achievable by very gentle lysing and washing of the red blood cells. Subsequently, it was found that the presence of very small traces residues of blood cell components such as endotoxins and phospholipids led to adverse reactions of the product in animal trials. Even after the product has been subjected to several diafiltration steps, it still contains unacceptably high traces of harmful impurities such as erythrocyte enzymes, modified and variant forms of hemoglobin, phospholipids and surface antigens.
One of the most urgent challenges in the blood substitute area, specifically the hemoglobin-based oxygen carriers, is the to develop processes for efficient and economical purification of proteins on a commercial scale. The need for a cost effective and efficient purification system is perhaps the most urgent need in this area, because the manufacture of large quantities of purified hemoglobin needs also to be able to meet the required cost criteria.
Whereas resolution and analysis time are important in analytical separations, the critical parameters in preparative chromatography, for commercial or semi-commercial scale use, are:
the amount of material isolated per unit time at a specific level of purity (throughput); and PA1 economics of the process, such as column sizes, media, buffers, equipment, recycle and re-use of components and reagents and the like. PA1 a) binding of the Hb at elevated pH, and elution with a descending pH gradient or step gradient of lower pH; PA1 b) binding of the Hb at high pH, low ionic strength and elution with a salt gradient; PA1 c) loading under pH conditions where the hemoglobin does not bind to the anion exchanger, but passes through the column unretained, while the impurities (more acidic contaminants) are captured on the column.
There are no reported methods for the purification of HbAo which meet the criteria for a cost efficient production process.
A hemoglobin-based blood substitute needs to be based either on a single hemoglobin form, or, if more than one form is present, a carefully controlled composition of known hemoglobin forms. Accordingly, a successful hemoglobin purification process needs to be capable of separating one hemoglobin form from another, as well as separating the desired Hb form from other contaminating red blood cells such as erythrocyte enzymes, phospholipids and surface antigens.
Chromatographic methods have been applied to the purification of hemoglobin solutions. U.S. Pat. No. 4,925,474 Hsia et al. describes the application of the techniques of affinity chromatography to hemoglobin purification, using columns in which a ligand showing preferential chemical binding affinity to the DPG site of hemoglobin was bound to the stationery phase of the column.
Ion exchange chromatographic techniques have also been applied to hemoglobin purification. The basic principles of the techniques of ion exchange chromatography are well known. A mixture of different species in a solution is applied to a suitably prepared ion exchange column. Each of the species in the mixture has a different affinity for the chemical reactant groups on the column. By varying the conditions on the column, e.g. the pH of the solution, the individual species can be arranged to bind or to elute from the column selectively, so as to separate the species individually from the mixture. The application of the technique to the purification of proteins such as hemoglobin is economically unattractive, except when used for small scale operations and analytical work. When hemoglobin is to be purified on a commercial scale, for use for example as an oxygen carrying rescusitative fluid (blood substitute), the technique as conventionally applied is impractical. The amounts of hemoglobin to be absorbed on and subsequently eluted from a chromatography column are so large that the column size requirements become impractically large and expensive.
Christensen et al., J. Biochem. Phys. 17 (1988), 143-154, reported the chromatographic purification of human hemoglobin. The methodology used represented a standard ion exchange chromatographic approach that did not provide opportunities for economical scale-up to production levels.
U.S. Pat. No. 5,084,588 Rausch and Feola (Biopure), describes standard anion and cation exchange chromatography methods for application to separation and purification of hemoglobin. In the case of anion exchange chromatography, three standard approaches are listed in this patent:
Approaches a) and b) have been extensively documented, but are not attractive for large scale production, owing to the limitation of low loading capacities necessary to achieve sufficient resolution of the hemoglobin products. These loading capacities are routinely only 20-30 mg/ml, which dictate prohibitively large and expensive columns for commercial scale purification of hemoglobin. For example, each 50 gm dose of final hemoglobin-based oxygen carrier (HBOC) would require a column of 1.5-2.5 liters.
Whilst approach c) would appear on the surface to be the most pragmatic, it turns out in practice that the chromatographic properties of human adult, normal, unmodified hemoglobin HbAo and some of the major contaminants such as HbAlc are not sufficiently distinct for practical application of this approach.
The standard approaches to cation exchange chromatography of mammalian hemoglobin have similar limitations.